The affinity maturation of anti-4-hydroxy-3-nitrophenylacetyl mouse monoclonal antibody. A calorimetric study of the antigen-antibody interaction

J Biol Chem. 1995 Sep 22;270(38):22218-22. doi: 10.1074/jbc.270.38.22218.


To understand the mechanism of affinity maturation, we examined the antigen-antibody interactions between 4-hydroxy-3-nitrophenylacetyl (NP) caproic acid and the Fab fragments of three anti-NP antibodies, N1G9, 3B44, and 3B62, by isothermal titration calorimetry. The analyses have revealed that all of these interactions are mainly driven by negative changes in enthalpy. The enthalpy changes decreased linearly with temperature in the range of 25-45 degrees C, producing negative changes in heat capacity. On the basis of the dependence of binding constants on the sodium chloride concentration, we have shown that, during the affinity maturation of the anti-NP antibody, the electrostatic effect does not significantly contribute to the increase in the binding affinity. We have found that, as the logarithm of the binding constants increases during the affinity maturation of the anti-NP antibody, the magnitudes of the corresponding enthalpy, heat capacity, and unitary entropy changes increase almost linearly. On the basis of this correlation, we have concluded that, during the affinity maturation of the anti-NP antibody, a better surface complementarity is attained in the specific complex in order to obtain a higher binding affinity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / immunology*
  • Antibody Affinity*
  • Binding Sites, Antibody
  • Calorimetry
  • Haptens
  • Immunoglobulin Fab Fragments / chemistry
  • In Vitro Techniques
  • Mice
  • Nitrophenols / chemistry
  • Nitrophenols / immunology*
  • Osmolar Concentration
  • Phenylacetates
  • Temperature
  • Thermodynamics


  • Antibodies, Monoclonal
  • Haptens
  • Immunoglobulin Fab Fragments
  • Nitrophenols
  • Phenylacetates
  • 4-hydroxy-5-nitrophenyl acetic acid